It has been of interest to determine the specific enzymes involved in the catabolism of endogenous opioid peptides and then, in turn, to develop selective inhibitors for these enzymes. Research on this aspect has been mostly on enkephalins but the metabolism of other endogenous opioid peptide is less well explored. Previously we have observed in vitro that met5-enkephalin-arg6-phe7 (ME-arg-phe) can be readily hydrolyzed by a dipeptidyl carboxypeptidase and, furthermore, this reaction can be effectively inhibited by captopril. In this study, whether the dipeptidyl carboxypeptidase participates in physiological inactivation of ME-arg-phe released was investigated using the in vivo superfusion of rat spinal cord. The release of opioid peptides, from spinal cord was induced by substance P. Without peptidase inhibitors, the efflux of ME-arg-phe and met5-enkephalin (ME) was hardly detectable. The inclusion of captopril in the perfusion medium increased the recovery of released ME-arg-phe but not that of ME. Combination of captopril and bestatin further increased ME-arg-phe release slightly. The recovery of ME released was greatly enhanced by bestatin but not by captopril or thiorphan (generally known as enkephalinase inhibitor). The results suggest that the dipeptidyl carboxypeptidase may play an important role in inactivation of released ME-arg-phe but not that of ME in vivo. Furthermore, the analgesic effect of ME-arg-phe applied intrathecally was greatly potentiated by captopril. Therefore it is highly probable that the dipeptidyl carboxypeptidase participate actively in the termination of ME-arg-phe activity in the spinal cord. The catabolism of ME released from spinal cord seems to be actively metabolized by aminopeptidase.